A fundamental property of a cell is the ability to regulate biochemical and genetic activities. One specific, but important, control mechanism is the phosphorylation-dephosphorylation reaction. The objective of this proposal is to apply an approach to study global changes as a result of signal transduction with phosphorylation cascades. Methodology employing affinity chromatography and micro-column liquid chromatography tandem mass spectrometry will be used to study the S. cerevisiae mating pheromone response pathway. This system has been partially characterized through genetic analysis, and has been found to be related to the MAPK family. Difference analysis of phosphopeptides produced by proteolytic digestion of affinity purified phosphoproteins from whole cell lysates of an activated cell line and a control cell line will be used to identify and characterize phosphorylation of proteins during signal transduction. The specific aims of the proposal are to: apply an approach using affinity chromatography and micro-column liquid chromatography tandem mass spectrometry to targets involved in biological control mechanisms and characterize the sites and level of phosphorylation of those targets. Global changes brought on by activation of the mating pheromone pathway will be identified with this method. It is anticipated that this approach will be applicable to any type of single cell organism or cell line for which perturbations of cellular physiology can be created. While the types of genetic experiments that can be performed on S. cerevisiae are not universal, other types of genetic manipulations can be performed. For example, homologous recombination can be used to ablate or alter genes and modify cellular phenotypes in mammalian cell lines. Thus, combining a technique for characterizing protein structure and modifications with genetic manipulations will significantly broaden the underlying knowledge of genetics, physiology, and phenotype.